Our goal is to understand the pathogenesis and host immune responses of otitis media and to develop a multivalent vaccine that reduces the incidence of otitis media (OM) caused by all major bacterial pathogens in children. For the passing year, we have worked on and/or finished the following four research projects. Lipooligosaccharide (LOS), a major bacterial component of the outer membrane of Moraxella catarrhalis or nontypeable Haemophilus influenzae, is a possible virulence factor in the pathogenesis of OM caused by the organisms. To elucidate the role of M. catarrhalis LOS in OM, we investigated the effects of LOS in matrix metalloprotease (MMPs) production and its signaling pathway in RAW 264.7 macrophage like cell line. The LOS dose dependently increased the production of MMP-9 in both RAW 264.7 cells and bone marrow derived macrophages (BMM&#966;). Inhibition of p38 and ERK1/2 completely inhibited LOS induced MMP-9 production while inhibition of JNK1/2 by SP600125 increased MMP-9 expression and production at both mRNA and protein levels by almost five fold. This result was confirmed by knocking down JNK1/2 by siRNA. We also observed the same phenomenon in BMM&#966;although kinetics was different from that of RAW 264.7 cells. In contrast to increased MMP-9 level, we found dose dependent inhibition of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) production by SP600125. SP600125 significantly increased both migration and invasion of RAW 264.7 cells. Therefore, JNK1/2 is suggested to negatively regulate LOS induced MMP-9 production in murine macrophages. The information further extends our understanding of the roles of LOS in the pathogenesis of OM. We previously identified two late acyltransferase genes lpxX and lpxL responsible for lipid A biosynthesis in M. catarrhalis because Lipid A moiety of the LOS is the key biological component of the LOS molecule. However, the interaction of both genes in the function of the M. catarrhalis in vitro and in vivo is unclear. Recently, a double knockout mutant O35ElpxXL in M. catarrhalis strain O35E was constructed to further study the biological interaction and roles of both genes. Structural analysis of lipid A moieties from the parental and mutant strains showed that the O35ElpxXL lacked three fatty acids: two decanoic acids (C10:0) and one dodecanoic (lauric) acid (C12:0). The resulting mutant was viable but with a reduced growth rate in broth culture. The mutant also presented significantly lower toxicity in the Limulus amebocyte lysate (LAL) assay, reduced resistance to normal human serum and to the hydrophobic reagents, reduced adherence to human epithelial cells, and accelerated bacterial clearance in a murine model of bacterial pulmonary clearance. When compared with either single mutant, the double mutant showed a dramatic reduction in toxicity and other biological activities, suggesting a synergic effect of both lpxX and lpxL genes. These results indicate that these two genes encoding late acyltransferases responsible for incorporation of the acyloxyacyl linked secondary acyl chains into the lipid A of the LOS molecule play important roles for biological activities and/or virulence of M. catarrhalis. Based on our previous experimental data, the M. catarrhalis LOS is not only a potential virulence factor but also a protective component that has been used as a successful vaccine candidate. In an effort to develop more conservative and safe LOS based vaccine candidates, LOS mutant strains O35Elgt5 and O35EgalE with one and two terminal galactose deletion of its LOS were constructed since a similarity between Gala1-4Gal1-4Glc structure on the M. catarrhalis LOS and human glycophingolipid antigens is implicated. Subsequently, their LOS-based protein conjugate vaccines were synthesized and characterized. The detoxified LOS (dLOS) component in the conjugate vaccines appeared in a more than 10,000-fold reduction of the toxicity when compared with that of the LOS. The antigenicity of the conjugates was similar to that of the corresponding LOS. Two subcutaneous immunizations with dLOS-BSA from O35E, O35Elgt5, or O35EgalE elicited a 6561-, 140-, or 27-fold rise of serum immunoglobulin G (IgG) in rabbits. Two subcutaneous immunizations with dLOS-TT from O35E, O35Elgt5, or O35EgalE elicited a 6561-, 1263-, or 243-fold rise of serum IgG in rabbits. In addition, rabbit antisera elicited by all conjugates presented elevated complement-mediated bactericidal activities against the wild type strain O35E. Furthermore, the rabbit antisera elicited by the O35Elgt5 conjugate vaccine showed better coverage in binding reactivity among M. catarrhalis pathogenic strains. The results indicate that the conjugate vaccines derived from mutants, especially O35Elgt5, can be used as a promising vaccine candidate for further investigation. OM can occur following outset of upper respiratory tract infections. Inhibition of bacterial colonization in nasopharynx by mucosal vaccination may prevent OM by reducing bacterial invasion of the middle ears. Previous studies have documented that the chinchilla model is a state-of-the-art model that mimics OM in humans and has been used for evaluating the efficacy of vaccine candidates by systemic vaccination. However, there is a lack of information as to whether the mucosal immunization would generate immunity against nontypeable H. influenzae nasopharynx colonization and subsequently prevent OM in a chinchilla model of OM. In the present study, 80 chinchillas were intranasally (i.n.) immunized with a dLOS-TT conjugate vaccine of nontypeable H. influenzae mixed with cholera toxin (CT) or CT. All vaccinated animals responded with elevated levels of mucosal and serum anti-LOS antibodies. Two weeks after the last immunization, 40 chinchillas were challenged i.n. with nontypeable H. influenzae to evaluate nasopharynx colonization while the rest of the animals were challenged transbullarly (T.B.) to examine the development of OM. Compared to the control group, the vaccination inhibited not only bacterial colonization in nasopharynx and transmission to middle ears in the i.n. challenge group but also bacterial colonization in nasopharynx and transmission to unchallenged ears in the T.B. challenge group. Current results along with our previous data indicate that mucosal vaccination is capable of inhibiting nontypeable H. influenzae colonization and preventing OM occurrence in chinchillas;the i.n. challenge model is preferable for testing the mucosal vaccines while the T.B. challenge model is superior for testing the systemic vaccines.